The overall goals of this proposal is to delineate the processes involved in the regulation of the synthesis and release of endothelins (ETs) in ocular tissues and to determine endothelin's target sites and cellular mechanism of action. The hypothesis to test is that ET is synthesized and stored in ciliary epithelium and other tissues and is released by a variety of signals, including cytokines to exert paracrine effects on the ciliary muscle and trabecular meshwork to enhance aqueous humor outflow and decrease intraocular pressure. Preliminary data from the applicant's laboratory indicates that the proinflammatory cytokine TNF-alpha stimulates the synthesis and release of ET-1 from ciliary epithelium and ET-1 acts on human ciliary muscle through an ETA receptor to enhance PLC activity and calcium mobilization. This latter effect is thought to be responsible for the muscle contraction induced by ET. Although endothelins' actions on intraocular pressure and contraction of the vascular smooth muscle are well documented, the mechanisms responsible for the regulation of ocular ET synthesis, release and actions of endothelins are not fully understood. The following specific aims are planned to address these mechanisms: (1) to determine if ET, its precursors and synthetic enzymes are present in human ciliary epithelium, ciliary muscle and trabecular meshwork cells and tissues using immunofluorescent microscopy, radioimmunoassay and Western Blot analysis; (2) to determine the signals and mechanisms responsible for the regulation of ET's synthesis and release by investigating the signal transduction pathways activated by TNF-alpha and autonomic agonists, including the role of protein kinase C (PKC) isoforms, nitric oxide (NO), and other messengers on the release of ET; (3) to determine tissue site receptors and cellular mechanism of action released ETs using RT- PCR, RNase protection analysis and in situ hybridization histochemistry under normal cytokine, and adrenergic/cholinergic stimulated conditions, and characterize the signal transduction pathways associated with ET receptor activation; and (4) to determine the functional role of released endothelins by linking the signal transduction mechanisms to contraction in the ciliary muscle and trabecular meshwork by measuring single cell contraction, myosin light chain phosphorylation, changes in outflow facility using the isolated anterior segment perfused human eye. Changes in ion transport process of the isolated ciliary epithelium will also be examined. These specific aims are designed to determine the mechanisms involved in the release and synthesis of ET in ocular tissues and to identify ET target sites. This study will provide information on the role ETs may play in intraocular pressure (IOP) homeostasis.